Methods for increasing circulating mesenchymal stem cells

ABSTRACT

Disclosed herein are methods for increasing circulating mesenchymal stem cells by administering a compound having the general formula 1 as defined herein, or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE

This application is a U.S. national phase of International ApplicationNo. PCT/US2018/045935, filed on Aug. 9, 2018, which claims priority toU.S. Provisional Application No. 62/546,256, filed Aug. 16, 2017, bothof which are incorporated by reference herein in their entirety.

FEDERAL FUNDING STATEMENT

This invention was made with government support under MD 140084, awardedby the (DoD) US Department of Defense. The government has certain rightsin the invention.

BACKGROUND

Mesenchymal stein cells (MSCs) are multipotent progenitor cells of themesoderm capable of differentiating into a number of different cellstypes including osteoblasts, chondrocytes, myocytes, and adipocytes,implicating their potential to regenerate bone, cartilage, muscle, andfat, respectively. MSCs also have potent anti-inflammatory andimmunomodulatory properties. As a result of both of these capacities,MSCs are under clinical development for wide range of conditionsincluding, but not limited to: myocardial infraction, graft versus hostdisease, diabetes, liver cirrhosis, spinal cord injury, osteoarthritis,Crohn's disease, multiple sclerosis, aplastic anemia, systemic lupuserythematosus, rheumatoid arthritis, Parkinson's disease, brain injury,and muscular dystrophy. In these clinical programs, MSCs are sourcedfrom fetal or adult tissue, potentially expanded in culture, andinjected or infused into the patient. When administered intravenously,MSCs become trapped in the lung limiting the systemic exposure. Anorally dosed agent that can stimulate to proliferation of MSCs andincrease circulating MSCs would represent an exciting alternativeapproach to accessing these therapeutically powerful cells.

SUMMARY

In one aspect methods for (a) increasing circulating mesenchymal steincells (MSC) in a subject, or (b) treating a subject having a disorderselected from the group consisting of myocardial infraction,cardiovascular disease, graft versus host disease, diabetes, livercirrhosis, liver disease, spinal cord injury, osteoarthritis, Crohn'sdisease, multiple sclerosis, aplastic anemia, rheumatoid arthritis,autoimmune disorders including alopecia areata, autoimmune hemolyticanemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), someforms of juvenile idiopathic arthritis, glomerulonephritis, Graves'disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura,myasthenia gravis, pemphigus/pemphigoid, pernicious anemia,polyarteritis nodosa, polymyositis, primary biliary, cirrhosis,psoriasis, scleroderma/systemic sclerosis, Sjögren's syndrome, someforms of thyroiditis, some forms of uveitis, vitiligo, granulomatosiswith polyangiitis (Wegener's), organ transplantation, organ rejection,Parkinson's disease, neurodegenerative disorders, amyotrophic lateralsclerosis, Alzheimer disease, brain injury, and inflammatory lungdisease, are disclosed, comprising administering to subject in needthereof an amount effective to treat the disorder of a compound havingthe general formula 1 or a pharmaceutically acceptable salt thereof:

wherein:

-   -   ring A is a five-membered or six-membered heteroaryl or        heterocyclyl ring containing either a combination of two        non-adjacent nitrogen or oxygen atoms, or a combination of three        or four nitrogen or oxygen atoms;    -   ring B is a five-membered or six-membered heteroaryl ring that        contains at least one nitrogen atom;    -   A¹, A², A³, A⁴ are independently selected from a group        consisting of ═N—, —C(═O)—, —C(R^(a))═, ═C(R^(b))—,        —C(R^(c))(R^(d))—N(R^(e))—, —C(R^(c))(R^(d))—O—, and        —[C(R^(c))(R^(d))]_(n)—, wherein n is 1 or 2;    -   X¹—X² is —(R⁶)C—N—, —N—C(R⁶)—, —N—N—, —N—O—, —O—N—, —N—S— or        —S—N—;    -   X³ is —(R⁷)C═C(R⁸)—, —O—, —S—, or —N(R⁹)—;    -   Z is —O—, —N(H)— or a bond to R⁵;    -   R^(a) and R^(b) are independently selected from the group        consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy,        aryloxy, formyl, acyl, acylamido and carboxy,    -   or R^(a) and R^(b) can also join to form a ring of up to 6        atoms;    -   R^(c) and R^(d) are independently selected from a group        consisting of hydrogen, alkyl, aryl, or heteroaryl, provided        that R^(c) and R^(d), together with the atoms to which they are        attached, form a ring of up to 6 atoms;    -   R^(e) is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl;    -   R¹, R³, R⁴, R⁶, R⁷, and R⁸ are independently selected from a        group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, arylmethyl, heteroarylmethyl, fluoro, chloro, bromo,        iodo, cyano, hydroxy, amino, alkylamino, alkoxy, aryloxy,        alkoxyalkyl and aryloxyalkyl;    -   R² is alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylmethyl,        heteroarylmethyl, alkoxy, trifluoromethoxy, perfluoroalkoxy,        aryloxy, alkoxyalkyl, or aryloxyalkyl;    -   R⁵ is alkyl, aryl, heteroaryl, hydroxyalkyl, carboxyalkyl,        alkoxyalkyl, or aryloxyalkyl; and    -   R⁹ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl.

In one embodiment, ring A is selected from the group consisting of:

wherein:

-   -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido and carboxy,    -   or R¹⁰ and R¹¹, together with ring A to which they are attached,        form a carbocyclic, heterocyclic, aryl or hetoaryl ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹³ is hydrogen, alkyl, aryl or heteroaryl;    -   R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl; and    -   R^(f), R^(g), R^(h), and R^(i), are independently selected from        a group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, arylmethyl, heteroarylmethyl, fluoro, chloro, bromo,        iodo, hydroxy, amino, alkylamino, alkoxy, aryloxy, alkoxyalkyl,        or aryloxyalkyl;    -   or a pharmaceutically acceptable salt thereof.

In another embodiment, ring B is selected from the group consisting of:

wherein groups R⁶, R⁷, R⁸ and R⁹ are defined as in general formula 1;

or a pharmaceutically acceptable salt thereof.

In a further embodiment, the compound is selected from the groupconsisting of:

wherein:

-   -   R¹, R², R³, R⁴ R⁵, R⁶, R⁷, R⁸, R⁹, R^(a), R^(b), R^(c), R^(d)        and Z are defined as in general formula 1;    -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido and carboxy    -   or R¹⁰ and R¹¹, together with ring A to which they are attached,        form a carbocyclic, heterocyclic, aryl or hetoaryl ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹³ is hydrogen, alkyl, aryl or heteroaryl;    -   R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl; and    -   R^(f), R^(g), R^(h), and R^(i), are independently selected from        a group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, arylmethyl, heteroarylmethyl, fluoro, chloro, bromo,        iodo, hydroxy, amino, alkylamino, alkoxy, aryloxy, alkoxyalkyl,        and aryloxyalkyl;    -   or a pharmaceutically acceptable salt thereof.

In various further embodiments, R² is trifluoromethoxy and/or Z is O or—N(H)—. In another embodiment, the compound has the general formula 4aor a pharmaceutically acceptable salt thereof:

wherein:

-   -   Z is —O— or —N(H)—;    -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido and carboxy,    -   or R¹⁰ and R¹¹, together with ring A to which they are attached,        form a carbocyclic, heterocyclic, aryl or hetoaryl ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹⁵ is alkyl, aryl, heteroaryl, arylmethyl, heteroarylmethyl,        trifluoromethyl or pentafluoroethyl; and    -   R¹⁶ is hydrogen, hydroxy, methoxy, alkoxy, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, amino, alkylamino, or dialkylamino.

In a further embodiment, the compound has the general formula 4a or apharmaceutically acceptable salt thereof:

wherein:

-   -   Z is —O— or —N(H)—;    -   R¹⁰, R¹¹ and R¹² are hydrogen;    -   R¹⁵ is alkyl, aryl, heteroaryl, arylmethyl, heteroarylmethyl,        trifluoromethyl or pentafluoroethyl; and    -   R¹⁶ is hydrogen, hydroxy, methoxy, alkoxy, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, amino, alkylamino, or dialkylamino.

In another embodiment, the compound has the general formula 4a or apharmaceutically acceptable salt thereof:

wherein:

-   -   Z is —O— or —N(H)—;    -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido or carboxy, provided that R¹⁰ and R¹¹ can also        be joined to form a carbocyclic, heterocyclic, aryl or hetoaryl        ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹⁵ is trifluoromethyl and R¹⁶ is ethyl

In a further embodiment, the compound has the formula 7 or apharmaceutically acceptable salt thereof:

DESCRIPTION OF THE FIGURES

FIG. 1 . 5 week-old wild-type (WT) and mdx mice were orally treated for6 weeks with either vehicle (Veh) or 8 mg/kg of Compound 7 once or twicedaily. (A) At necropsy, bone marrow was collected, cultured, and CFU-Fcounts were recorded which showing a significant increase in MSC withboth once and twice daily treatment with Compound 7. (B) The percentageof CD45 negative cells triply positive for MSC markers Sca-1, CD29, &CD105 from the collected bone marrow was analyzed by flow cytometry.Relative to vehicle treated mdx mice, mdx mice treated with Compound 7had significantly increased percentage of CD45 negative cells triplypositive for Sca-1, CD29, & CD105 to levels comparable to vehicletreated wild-type mice. (C) The percentage of CD45 negative cells triplypositive for MSC markers Sca-1, CD29, & CD105 from the peripheral bloodwas analyzed by flow cytometry showing a significant increase incirculating MSCs with twice a day oral treatment with Compound 7.*=P≤0.05, **=P≤0.01, and ***=P≤0.001 in comparison with vehicle control.

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

PCT Application PCT/US14/30071 provided novel non-peptidic compounds andcompositions (including the synthesis thereof) capable of modulation theMas receptor of the Renin-Angiotensin System (RAS) and/or capable ofmimicking, in part or in entirety, the in vitro and in vivo activitiesof the endogenous Mas receptor ligand A (1-7).

The present invention describes the use of compounds and compositionsfor the increasing circulating mesenchymal stein cells (MSC), and thususe of the compounds for treating a variety of disorders that canbenefit from increasing circulating MSC, including but not limited tomyocardial infraction, graft versus host disease, diabetes, livercirrhosis, spinal cord injury, osteoarthritis, Crohn's disease, multiplesclerosis, aplastic anemia, rheumatoid arthritis, Parkinson's disease,and brain injury.

In various embodiments, this invention provides methods for

-   -   (a) treating a subject having a disorder selected from the group        consisting of myocardial infraction, cardiovascular disease,        graft versus host disease, diabetes, liver cirrhosis, liver        disease, spinal cord injury, osteoarthritis, Crohn's disease,        multiple sclerosis, aplastic anemia, rheumatoid arthritis,        autoimmune disorders including alopecia areata, autoimmune        hemolytic anemia, autoimmune hepatitis, dermatomyositis,        diabetes (type 1), some forms of juvenile idiopathic arthritis,        glomerulonephritis, Graves' disease, Guillain-Barré syndrome,        idiopathic thrombocytopenic purpura, myasthenia        pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,        polymyositis, primary biliary, cirrhosis, psoriasis,        scleroderma/systemic sclerosis, Sjögren's syndrome, some forms        of thyroiditis, some forms of uveitis, vitiligo, granulomatosis        with polyangiitis (Wegener's)), organ transplantation, organ        rejection, Parkinson's disease, neurodegenerative disorders,        amyotrophic lateral sclerosis, Alzheimer disease, brain injury,        and inflammatory lung disease, comprising administering to        subject in need thereof an amount effective to treat the        disorder of a compound having the general formula 1 including        salts thereof; or    -   (b) increasing circulating mesenchymal stem cells (MSC) in a        subject, comprising administering to a subject in need thereof        an amount effective to increase circulating MSC in the subject        of a compound having the general formula 1 or a pharmaceutically        acceptable salt thereof:

wherein:

-   -   ring A is a five-membered or six-membered heteroaryl or        heterocyclyl ring containing either a combination of two        non-adjacent nitrogen or oxygen atoms, or a combination of three        or four nitrogen or oxygen atoms;    -   ring B is a five-membered or six-membered heteroaryl ring that        contains at least one nitrogen atom;    -   A¹, A², A³, A⁴ are independently selected from a group        consisting of ═N—, —C(═O)—, —C(R^(a))═, ═C(R^(b))—,        —C(R^(c))(R^(d))—N(R^(e))—, —C(R^(c))(R^(d))—O—, and        —[C(R^(c))(R^(d))]_(n)—, wherein n is 1 or 2;    -   X¹—X² is —(R⁶)C—N—, —N—C(R⁶)—, —N—N—, —N—O—, —O—N—, —N—S— or        —S—N—;    -   X³ is —(R⁷)C═C(R⁸)—, —O—, —S—, or —N(R⁹)—;    -   Z is —O—, —N(H)— or a bond to R⁵;    -   R^(a) and R^(b) are independently selected from the group        consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy,        aryloxy, formyl, acyl, acylamido and carboxy,    -   or R^(a) and R^(b) can also join to form a ring of up to 6        atoms;    -   R^(c) and R^(d) are independently selected from a group        consisting of hydrogen, alkyl, aryl, or heteroaryl, provided        that R^(c) and R^(d), together with the atoms to which they are        attached, form a ring of up to 6 atoms;    -   R^(e) is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl;    -   R¹, R³, R⁴, R⁶, R⁷, and R⁸ are independently selected from a        group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, aryl methyl, heteroarylmethyl, fluoro, chloro,        bromo, iodo, cyano, hydroxy, amino, alkylamino, alkoxy, aryloxy,        alkoxyalkyl and aryloxyalkyl;    -   R² is alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylmethyl,        heteroarylmethyl, alkoxy, trifluoromethoxy, perfluoroalkoxy,        aryloxy, alkoxyalkyl, or aryloxyalkyl;    -   R⁵ is alkyl, aryl, heteroaryl, hydroxyalkyl, carboxyalkyl,        alkoxyalkyl, or aryloxyalkyl; and    -   R⁹ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl.    -   In exemplary embodiments, ring A includes but is not limited to        a ring selected from a group consisting of:

wherein:

-   -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido or carboxy, provided that R¹⁰ and R¹¹ can also        be joined to form a carbocyclic, heterocyclic, aryl or hetoaryl        ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹³ is hydrogen, alkyl, aryl or heteroaryl;    -   R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl; and    -   R^(f), R^(g), R^(h), and R^(i), are independently selected from        a group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, arylmethyl, heteroarylmethyl, fluoro, chloro, bromo,        iodo, hydroxy, amino, alkylamino, alkoxy, aryloxy, alkoxyalkyl,        or aryloxyalkyl.

In other exemplary embodiments, ring B includes but is not limited to afive- or six-membered heteroaryl ring selected from a group consistingof:

wherein groups R⁶, R⁷, R⁸ and R⁹ are defined as in general formula 1

In some exemplary embodiments, the compounds administered in connectionwith the methods and compositions provided herein have the generalformula selected from a group consisting of:

wherein groups R¹, R², R³, R⁴ R⁵, R⁶, R⁷, R⁸, R⁹, A¹, A², A³, A⁴ and Zare defined as in general formula 1.

In other exemplary embodiments, the compounds have the general formulaselected from a group consisting of:

wherein:

-   -   R¹, R², R³, R⁴ R⁵, R⁶, R⁷, R⁸, R⁹, R^(a), R^(b), R^(c), R^(d)        and Z are defined as in general formula 1.    -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido or carboxy, provided that R¹⁰ and R¹¹ can also        be joined to form a carbocyclic, heterocyclic, aryl or hetoaryl        ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹³ is hydrogen, alkyl, aryl or heteroaryl;    -   R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl; and    -   R^(f), R^(g), R^(h), and R^(i), are independently selected from        a group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, arylmethyl, heteroarylmethyl, fluoro, chloro, bromo,        iodo, hydroxy, amino, alkylamino, alkoxy, aryloxy, alkoxyalkyl,        or aryloxyalkyl.

In additional exemplary embodiments, the compounds administered inconnection with the methods and compositions provided herein have thegeneral formula selected from a group consisting of:

wherein:

-   -   R¹, R², R³, R⁴ R⁵, R⁶, R⁷, R⁸, R⁹, R^(a), R^(b), R^(c), R^(d)        and Z are defined as in general formula 1.    -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido or carboxy, provided that R¹⁰ and R¹¹ can also        be joined to form a carbocyclic, heterocyclic, aryl or hetoaryl        ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹³ is hydrogen, alkyl, aryl or heteroaryl;    -   R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl; and    -   R^(f), R^(g), R^(h), and R^(i), are independently selected from        a group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, arylmethyl, heteroarylmethyl, fluoro, chloro, bromo,        iodo, hydroxy, amino, alkylamino, alkoxy, aryloxy, alkoxyalkyl,        or aryloxyalkyl.

In some preferred embodiments, the compounds administered in connectionwith the methods provided herein have the general formula 2a,b or 3a,b:

wherein:

-   -   R¹, R², R³, R⁴ R⁵, R⁶, R⁷, R⁸, R⁹, R^(a), R^(b), R^(c), R^(d)        and are defined as in general formula 1.    -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido or carboxy, provided that R¹⁰ and R¹¹ can also        be joined to form a carbocyclic, heterocyclic, aryl or hetoaryl        ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹³ is hydrogen, alkyl, aryl or heteroaryl;    -   R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl; and    -   R^(f), R^(g), R^(h), and R¹, are independently selected from a        group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, arylmethyl, heteroarylmethyl, fluoro, chloro, bromo,        iodo, hydroxy, amino, alkylamino, alkoxy, aryloxy, alkoxyalkyl,        or aryloxyalkyl.

In further preferred embodiments the compounds administered inconnection with the methods and compositions provided herein having thegeneral formula 4a,b, 5a,b or 6a,b:

wherein:

-   -   R¹, R², R³, R⁴ R⁵, R⁶, R⁷, R⁸, R⁹, R^(a), R^(b), R^(c), R^(d)        and Z are defined as in general formula 1.    -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido or carboxy, provided that R¹⁰ and R¹¹ can also        be joined to form a carbocyclic, heterocyclic, aryl or hetoaryl        ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹⁴ is hydrogen, alkyl, aryl, heteroaryl, acyl, alkoxyacyl,        aminoacyl, dialkylaminoacyl, or dialkylaminoacyl; and    -   R¹⁵ is alkyl, aryl, heteroaryl, arylmethyl, heteroarylmethyl,        trifluoromethyl or pentafluoroethyl; and    -   R¹⁰ is hydrogen, hydroxy, methoxy, alkoxy, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, amino, alkylamino, or dialkylamino.

In some exemplary embodiments, the R¹⁰, R¹¹ and R¹² are hydrogen, andR¹⁴ is methyl.

In other exemplary embodiments, R¹⁵ is trifluoromethyl and R¹⁶ is ethyl.

Preferred embodiments of the compounds administered in connection withthe methods and compositions provided herein have the general formula4a:

wherein:

-   -   Z is O or —N(H)—;    -   R¹⁰ and R¹¹ are independently selected from a group consisting        of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, halo,        hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, formyl,        acyl, acylamido or carboxy, provided that R¹⁰ and R¹¹ can also        be joined to form a carbocyclic, heterocyclic, aryl or hetoaryl        ring;    -   R¹² is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl,        halo, hydroxy, hydroxyalkyl, alkoxyalkyl, alkoxy, aryloxy, or        acylamido;    -   R¹⁵ is alkyl, aryl, heteroaryl, arylmethyl, heteroarylmethyl,        trifluoromethyl or pentafluoroethyl; and    -   R¹⁶ is hydrogen, hydroxy, methoxy, alkoxy, alkyl, alkenyl,        alkynyl, aryl, heteroaryl, amino, alkylamino, or dialkylamino.

In some exemplary embodiments, the R¹⁰, R¹¹ and R¹² are hydrogen.

In exemplary embodiments, R¹⁵ is trifluoromethyl and R¹⁶ is ethyl.

Exemplary embodiments of compounds administered in connection with themethods provided herein are provided by compounds 7, 8, 9, 10, and 11:

A representative exemplary embodiment of the provided methods disclosedherein comprises the administration of Compound 7:

The invention further provides pharmaceutical compositions for themethods of the invention, comprising a compound provided in PCTApplication PCT/US14/30071 or a pharmaceutically acceptable salt, and apharmaceutically acceptable carrier as described in PCT ApplicationPCT/US14/30071. The provided methods and compositions are employed inany suitable administrative form, including but not limited to oral,parenteral, or topical administration.

The invention is further described in the attached examples, which areillustrative only, and which are not intended to limit the scope of theinvention described in the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. In the event that there is a plurality of definitions for aterm herein, those in this section will control unless stated otherwise.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be therapeutic in terms of a partial or complete cure for adisease and/or adverse symptoms attributable to the disease.“Treatment”, as used herein, covers any treatment of the reciteddisorders, particularly in a human, and includes: (a) limitingdevelopment of symptoms or flares from occurring in a subject having thedisorder; (b) limiting worsening of symptoms or flares in a subjecthaving the disorder; (c) inhibiting the disorder, i.e., arrestingdevelopment of the disorder; (d) relieving the disorder, i.e., causingregression of the disorder.

Increasing circulating mesenchymal stem cells (MSC) as used herein maymean any increase over control (such as a subject not treated with thecompounds of the invention), as any such increase will provide atherapeutic benefit to the subject. In various non-limiting embodiments,the methods increase circulating MSC in the subject at least 5% comparedto control; in other embodiments, at least 10%, 15%, 20%, 25%, or morecompared to control.

The terms “individual,” “subject,” and “patient,” used interchangeablyherein, refer to a mammal, including, but not limited to, murines,simians, humans, mammalian farm animals, mammalian sport animals, andmammalian pets. Preferably, the subject herein is human, such as a humanfemale.

As used herein, the nomenclature alkyl, alkoxy, carbonyl, etc. is usedas is generally understood by those of skill in the chemical art. Asused in this specification, alkyl groups can include straight-chained,branched and cyclic alkyl radicals containing up to about 20 carbons, or1 to 16 carbons, and are straight or branched. Exemplary alkyl groupsherein include, but are not limited to, methyl, ethyl, propyl,isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl,neopentyl, tert-pentyl and isohexyl. As used herein, lower alkyl referto carbon chains having from about 1 or about 2 carbons up to about 6carbons. Suitable alkyl groups may be saturated or unsaturated. Further,an alkyl may also be substituted one or more times on one or morecarbons with substituents selected from a group consisting of C1-C15alkyl, allyl, allenyl, alkenyl, C3-C7 heterocycle, aryl, halo, hydroxy,amino, cyano, oxo, thio, alkoxy, formyl, carboxy, carboxamido,phosphoryl, phosphonate, phosphonamido, sulfonyl, alkylsulfonate,arylsulfonate, and sulfonamide. Additionally, an alkyl group may containup to 10 heteroatoms, in certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8 or9 heteroatom substituents. Suitable heteroatoms include nitrogen,oxygen, sulfur and phosphorous.

As used herein, “cycloalkyl” refers to a mono- or multicyclic ringsystem, in certain embodiments of 3 to 10 carbon atoms, in otherembodiments of 3 to 6 carbon atoms. The ring systems of the cycloalkylgroup may be composed of one ring or two or more rings which may bejoined together in a fused, bridged or spiro-connected fashion.

As used herein, “aryl” refers to aromatic monocyclic or multicyclicgroups containing from 3 to 16 carbon atoms. As used in thisspecification, aryl groups are aryl radicals, which may contain up to 10heteroatoms, in certain embodiments, 1, 2, 3 or 4 heteroatoms. An arylgroup may also be optionally substituted one or more times, in certainembodiments, 1 to 3 or 4 times with an aryl group or a lower alkyl groupand it may be also fused to other aryl or cycloalkyl rings. Suitablearyl groups include, for example, phenyl, naphthyl, tolyl, imidazolyl,pyridyl, pyrroyl, thienyl, pyrimidyl, thiazolyl and furyl groups.

As used in this specification, a ring is defined as having up to 20atoms that may include one or more nitrogen, oxygen, sulfur orphosphorous atoms, provided that the ring can have one or moresubstituents selected from a group consisting of hydrogen, alkyl, allyl,alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro,hydroxy, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino,acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio,alkylsulfonate, arylsulfonate, phosphoryl, phosphonate, phosphonamido,and sulfonyl, and further provided that the ring may also contain one ormore fused rings, including carbocyclic, heterocyclic, aryl orheteroaryl rings.

The term “alkenyl” refers to a branched or unbranched hydrocarbon havingat least one carbon-carbon double bond.

The term “alkynyl” refers to a branched or unbranched hydrocarbon havingat least one carbon-carbon triple bond.

The term “carboxy” refers to a —CO₂H group.

The term “hydroxy” refers to an —OH group.

The term “alkoxy” refers to a group of the formula R—O— where R is an“alkyl” as defined herein.

The term “carbocycle” refers to a non-aromatic stable 3- to 8-memberedcarbon ring which may be saturated, mono-unsaturated orpoly-unsaturated.

The term “amino” includes primary, secondary or tertiary amino groups.

The term “cyano” refers to the group —CN.

As used herein, alkenyl and alkynyl carbon chains, if not specified,contain from 2 to 20 carbons, or 2 to 16 carbons, and are straight orbranched. Alkenyl carbon chains of from 2 to 20 carbons, in certainembodiments, contain 1 to 8 double bonds, and the alkenyl carbon chainsof 2 to 16 carbons, in certain embodiments, contain 1 to 5 double bonds.Alkynyl carbon chains of from 2 to 20 carbons, in certain embodiments,contain 1 to 8 triple bonds, and the alkynyl carbon chains of 2 to 16carbons, in certain embodiments, contain 1 to 5 triple bonds.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system, in certain embodiments, of about 4 to about 15members where one or more, in one embodiment 1 to 4, of the atoms in thering system is a heteroatom, that is, an element other than carbon,including but not limited to, nitrogen, oxygen or sulfur. The heteroarylgroup may be optionally fused to a benzene ring. Heteroaryl groupsinclude, but are not limited to, furyl, imidazolyl, pyrrolidinyl,pyrimidinyl, triazolyl, tetrazolyl, thienyl, pyridyl, pyrrolyl,N-methylpyrrolyl, quinolinyl and isoquinolinyl.

As used herein, “heterocyclyl” refers to a monocyclic or multicyclicnon-aromatic ring system, in one embodiment of 3 to 10 members, inanother embodiment of 4 to 7 members, in a further embodiment of 5 to 6members, where one or more, in certain embodiments, 1 to 3, of the atomsin the ring system is a heteroatom, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen or sulfur. Inembodiments where the heteroatom(s) is(are) nitrogen, the nitrogen isoptionally substituted with alkyl, alkenyl, alkynyl, aryl, heteroaryl,aralkyl, heteroaralkyl, cycloalkyl, heterocyclyl, cycloalkylalkyl,heterocyclylalkyl, acyl, guanidino, or the nitrogen may be quaternizedto form an ammonium group where the substituents are selected as above.

As used herein, “aralkyl” refers to an alkyl group in which one of thehydrogen atoms of the alkyl is replaced by an aryl group.

As used herein, “halo”, “halogen” or “halide” refers to F, Cl, Br or I.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by halogen. Such groups include,but are not limited to, chloromethyl and trifluoromethyl.

As used herein, “aryloxy” refers to RO—, in which R is aryl, includinglower aryl, such as phenyl.

As used herein, “acyl” refers to a —COR group, including for examplealkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, or heteroarylcarbonyls,all of which may be optionally substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem.11:942-944).

As used herein, pharmaceutically acceptable derivatives of a compoundinclude salts, esters, enol ethers, enol esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydratesor prodrugs thereof. Such derivatives may be readily prepared by thoseof skill in this art using known methods for such derivatization. Thecompounds produced may be administered to animals or humans withoutsubstantial toxic effects and either are pharmaceutically active or areprodrugs. Pharmaceutically acceptable salts include, but are not limitedto, amine salts, such as but not limited to N,N-dibenzylethylenediamine,chloroprocaine, choline, ammonia, diethanolamine and otherhydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine,N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethylbenzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to lithium, potassium andsodium; alkali earth metal salts, such as but not limited to barium,calcium and magnesium; transition metal salts, such as but not limitedto zinc; and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates; and salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates. Pharmaceuticallyacceptable esters include, but are not limited to, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl andheterocyclyl esters of acidic groups, including, but not limited to,carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids,sulfinic acids and boronic acids. Pharmaceutically acceptable enolethers include, but are not limited to, derivatives of formula C═C(OR)where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,heteroaralkyl, cycloalkyl or heterocyclyl. Pharmaceutically acceptableenol esters include, but are not limited to, derivatives of formulaC═C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, aryl,heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl.Pharmaceutically acceptable solvates and hydrates are complexes of acompound with one or more solvent or water molecules, or 1 to about 100,or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.

EXAMPLES

Summary: We show herein that small molecule Mas receptor agonistsincrease circulating mesenchymal stem cells (MSCs). We have showndecreased bone marrow MSCs of two chronic inflammatory diseases,diabetes and Duchenne muscular dystrophy (DMD), relative to theirwild-type, non-disease control. Disclosed herein is the discovery thatMas agonists induce an increase in the therapeutically valuablecirculating MSCs, as measured by flow cytometry in the peripheral blood.

DMD is one of the most common and devastating genetic diseases ofchildhood caused by mutations in the DMD gene resulting in the loss offunctional dystrophin protein. In unaffected individuals, dystrophinserves a stabilizing role in muscle cells including cardiomyocytes. Dueto dystrophin deficiency, the muscle cells in DMD patients arepredisposed to contraction-induced damage. Over time, successive cyclesof injury and repair lead to a state of chronic inflammation, oxidativestress, and fibrosis which reduces the regenerative capacity of themuscle and drains satellite cell pools.

In the mdx mouse model of DMD, 5 week-old wild-type (C57BL/10SnJ) andmdx (C57BL/10ScSn-Dmd^(mdx)/J) mice were treated for 6 weeks andnecropsied. These mice were broken into 4 orally dosed groups: awild-type group (n=5) dosed twice daily with vehicle, an mdx group (n=8)dosed twice daily with vehicle, an mdx group (n=8) dosed once daily with8 mg/kg of Compound 7, and an mdx group (n=8) dosed twice daily with 8mg/kg of Compound 7. The vehicle was a 1% β-cyclodextrin in ˜pH 3citrate buffer and the treatment 1% β-cyclodextrin in ˜pH 3 citratebuffer with Compound 7. At necropsy, the femur bone marrow was isolatedand peripheral blood was harvested. The bone marrow was cultured inMesencult™ MSC Basal Medium (mouse) supplemented with Mesencult™ MSCStimulatory Supplement (Stemcell Technologies). CFU-F colonies of ≥50cells were counted after 8 days in culture. Daily oral treatment withCompound 7 significantly increased bone marrow MSCs with the twice dailydosing restoring levels comparable to the non-dystrophic controls (FIG.1A).

Assessment of MSCs by flow cytometry focused on non-hematopoietic cells(CD45 negative) that were positive for mouse MSC markers Sca-1 (stemcell antigen-1), CD29 (integrin β₁), and CD105 (endoglin). Analysis ofthe bone marrow showed a similar significant increase in the percentagecells triply positive for MSC markers (Sca-1, CD29, & CD105) innon-hematopoietic cells (CD45 negative) in mdx mice treated withCompound 7 over mdx mice treated with vehicle. In fact, Compound 7treatment restored the percentage of these triply positive cells tolevels comparable to non-dystrophic controls (FIG. 1B). However,unexpectedly, in the peripheral blood, twice daily oral treatment withCompound 7 resulted in significantly higher percentages ofnon-hematopoietic cells triply positive for MSC markers than both mdxand wild-type mice treated with vehicle, illustrating the ability ofthis representative small molecule Mas agonist at increasing circulatingMSCs (FIG. 1C).

Method

At necropsy, the femurs were also collected in a 1.5 mL microcentrifugetube containing ˜800 μL of sterile Dulbecco's phosphate-buffered saline(DPBS) with 2% fetal bovine serum (FBS) and 2× Penicillin (Pen) and 2×Streptomycin (Strep) (DPBS/2% FBS/2× Pen/Strep) and stored on ice. In abiosafety cabinet, both femurs from each mouse were cleaned of musclewith gauze, cut at both ends, and flushed into a 5 mL culture tubes with˜3 mL of sterile DPBS/2% FBS/2× Pen/Strep in a 3 mL syringe fitted witha 25G needle. The tubes were spun at 1,000 RPM for 10 minutes at 4° C.,resulting in a pellet. The supernatant was discarded and the marrow wasresuspended with 1 mL of sterile DPBS with 2% FBS and 2× Pen/Strep. Thecell solutions were counted with a Z1 Coulter Counter (Beckman Coulter)and resuspended at 5*10⁶ cells/mL. Two mL of Mesencult™ MSC Basal Medium(mouse) (Stemcell Technologies) supplemented with Mesencult™ MSCStimulatory Supplement (mouse) (Stemcell Technologies) were added toeach well of a 24 well plate along with 100 μL of cells from eachrespective sample. The plates were incubated for 8 days at 37° C. and 5%CO₂. On day 8, the colonies of ≥50 were counted by light microscopy.

During the necropsy, the blood was collected by cardiac puncture with a1 mL syringe fitted with a 22G needle, transferred into 2 ml K3E K3EDTAVACUETTE® tubes, and stored on ice. The tubes were then spun at 1,500RPM for 15 min at 4° C. The plasma (top layer) was collected. Theremaining pellet was resuspended with 500 μL of sterile DPBS/2% FBS/2×Pen/Strep, layered over 1 mL of Ficol-Paque™ PLUS (GE Healthcare) in a 5mL culture tube, and centrifuged at 1,200 RPM for 30 min at 4° C. withno brake. The resulting huffy coat was then collected with a 200 μLpipette, placed in a 5 mL culture tube, and washed (2 mL of sterileDPBS/2% FBS/2× Pen/Strep, centrifuged at 1,200 RPM for 5 min at 4° C.and supernatant decanted). The pellet was resuspended in 0.5 mL ofsterile DPBS/2% FBS/2× Pen/Strep. The cell solutions were counted with aZ1 Coulter Counter (Beckman Coulter) and resuspended at 5*10⁶ cells/mL.400 μL of the 5*10⁶ cells/mL solution was added to a 5 mL culture tube,the tube was centrifuge at 1,200 RPM at 4° C. for 15 minutes, and thesupernatant removed. The pellet was re-suspend in 50 μL of blockingbuffer (1 part Fc block and 10 parts DPBS/2% FBS) and incubated at 4° C.for 15 minutes. To this solution was added 50 μL of antibody cocktail (5μL of CD105-FITC (clone: MJ7/18), CD45-PerCP (clone: 30F11), CD29-PE(clone: HMβ1-1), and Anti-Sca-1-APC (clone: D7), and 30 μL of DPBS/2%FBS) which was incubated at 4° C. for 30 minutes. After this time, 3 mLof cold PBS was added and the tube was centrifuge at 1,200 RPM at 4° C.for 15 min. The supernatant was then removed, re-suspend in 500 μL 10%neutral buffered formalin, covered with aluminum foil, and stored at 4°C. until analyzed. Samples were then read on a LSR II flow cytometer (BDBiosciences, San Jose, Calif.). Data was analyzed using FlowJo™ V10.0.7r2.

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What is claimed is:
 1. A method for increasing circulating mesenchymalstem cells (MSC) in a subject, comprising administering to a subject inneed thereof an amount effective to increase circulating MSC to atherapeutically valuable level in the subject of a compound of formula7, 8, 9, 10, or 11 or a pharmaceutically acceptable salt thereof:

wherein the subject has a disorder selected from the group consisting ofosteoarthritis, aplastic anemia, autoimmune hemolytic anemia,dermatomyositis, Graves' disease, idiopathic thrombocytopenic purpura,pemphigus/pemphigoid, polyarteritis nodosa, psoriasis, Sjögren'ssyndrome, granulomatosis with polyangiitis, and organ rejection, andwherein the method serves to treat the disorder.
 2. The method of claim1, wherein the compound has the formula 7, or a pharmaceuticallyacceptable salt thereof:


3. The method of claim 1, wherein the provided compound orpharmaceutically acceptable salt thereof is provided as a compositioncomprising the compound or pharmaceutically acceptable salt thereof anda pharmaceutically acceptable carrier suitable for oral, parenteral, ortopical administration.
 4. The method of claim 1, wherein the subjecthas osteoarthritis or aplastic anemia.
 5. The method of claim 1, whereinthe subject has Graves' disease.
 6. The method of claim 1, wherein thesubject is also administered one or more other bone marrow/stem cellstimulating agents.
 7. The method of claim 6, wherein the bonemarrow/stem cell stimulating agent is selected from a CXCR4 antagonist,plerixafor, vascular endothelial growth factor, granulocyte colonystimulating factor, filgrastim, pegfilgrastim, erythropoietin, anddarbepoetin.